The canonical Wnt pathway regulates the constitutive level and intracellular localization of β-catenin, a key component of a tightly regulated receptor-mediated signal transduction network required for both embryonic development and adult tissue homeostasis. In unstimulated normal cells, β-catenin binds to adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), and Axin, which form a destruction complex that phosphorylates β-catenin, targeting it for proteosomal degradation. The binding of Wnt ligands to the frizzled and low-density lipoprotein receptors (LRP5 and LRP6) inhibits the activity of the GSK3β/APC/Axin complex, enabling non-phosphorylated β-catenin to undergo nuclear translocation to exert its transcriptional effects. Nuclear β-catenin associates with the lymphoid enhancer factor/T-cell factor (LEF/TCF) family of transcription factors to induce the expression of cell proliferation, migration, and survival genes, such as c-Myc and cyclin D1. Normally, this transcriptional pathway is turned off when Wnt ligands uncouple from their receptors. However, a variety of loss of function mutations in APC and Axin, and activating mutations in β-catenin itself, enable β-catenin to escape the destruction complex, persist in the nucleus, and drive oncogenic transcription.
In Drosophila melanogaster, the transcriptional activity of β-catenin further depends on two co-factors, BCL9 and Pygopus. The formation of a quaternary complex consisting of TCF, β-catenin, BCL9, and Pygopus enhances β-catenin-dependent Wnt transcriptional activity. The human BCL9 gene was first identified by cloning the t(1; 14)(q21; q32) translocation from a patient with precursor B-cell acute lymphoblastic leukemia (ALL). Amplifications of the chromosome 1q21-locus in which the BCL9 gene resides is observed in a broad range of human cancer types and it has been associated with tumor progression, decreased survival and poor clinical outcome. Most recently, insertional mutagenesis by the PiggyBac transposon has identified a hit in BCL9. Whereas in colorectal cancer (CRC) established mutations in APC and β-catenin drive the oncogenic phenotype, in multiple myeloma (MM) no such mutations have been reported and Wnt activation is instead driven by BCL9, implicating this β-catenin co-factor as a bona-fide oncogene. BCL9 overexpression has since been identified in a large subgroup of human tumors, yet is not expressed in the normal cellular counterparts from which the tumors originate. BCL9-mediated enhancement of β-catenin's transcriptional activity increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting the loss of an epithelial phenotype and gain of a mesenchyme-like functionality. shRNA-induced downregulation of BCL9 in vivo suppresses the expression of Wnt targets c-Myc, cyclin D1, CD44, and VEGF, and correspondingly increases the survival of xenograft mice with CRC and MM by reducing tumor load, metastasis, and the host angiogenesis response. The striking BCL9 dependence of these cancers and the expression of BCL9 in ˜30% of epithelial tumors provides a compelling rationale for targeting the BCL9/β-catenin protein interaction. Importantly, Bcl9-null mice lack an overt disease phenotype, suggesting that pharmacologic blockade of the BCL9/β-catenin complex may be relatively non-toxic.
The Wnt pathway consists of a tightly regulated receptor-mediated signal transduction system required for both embryonic development and adult tissue homeostasis in vertebrates and invertebrates and involves canonical and non-canonical Wnt pathways. Several components of the canonical Wnt signaling cascade have been shown to function as either tumor suppressor genes (TSG) or as oncogenes in a wide range of common human cancers including colorectal, hepatocellular, breast, endometrial carcinomas and MM. Furthermore, the canonical Wnt pathway has been implicated in the regulation of normal (e.g., wound healing) as well as pathological processes (e.g., diabetes). These observations underscore the relevance of this pathway to oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy, wound healing, angiogenesis and diabetes.
It is an object of the invention to design and generate hydrocarbon-stapled peptides of the HD2 domain of BCL9 (stapled α-helices of BCL9 or SAH-BCL9) to block Wnt signaling. It is also an object to demonstrate that direct binding of stabilized α-helical peptides to β-catenin prevents β-catenin/BCL9 interaction, Wnt transcriptional activity, and expression of downstream targets. Such mechanisms would result in a method of treatment of cancer cells with SAH-BCL9 and result in inhibition of tumor cell proliferation, migration, tumor-induced angiogenesis, tumor load, de-differentiation (epithelial-mesenchymal transition [EMT]), and metastasis in Wnt/β-catenin-driven cancers.